Differntiation method for production of glial cell population

ABSTRACT

The present invention provides methods for generating oligodendrocyte progenitor cells from pluripotent cells, as well as methods for sustaining these oligodendrocyte progenitor cells in relatively pure cultures for long periods of time. The present invention also provides methods for further differentiating these oligodendrocyte progenitor cells into various glial cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/406,664, filed Oct. 26, 2010 as is incorporatedherein by reference.

STATEMENT OF GOVERNMENT SPONSORED RESEARCH

This invention was supported in part by grant R21MH087877-01 from theNational Institute of Mental Health (NIMH). The Federal Government hascertain rights in this invention.

FIELD OF THE INVENTION

This invention relates to the production of cells and homogeneous cellpopulations of glial lineage.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

Effective conduction of action potentials in the mammalian centralnervous system (CNS) requires proper ensheathment and insulation ofneuron axons by myelin. Impairments of oligodendrocyte cells, themyelinogenic cells of the mammalian CNS, cause a number of debilitatingand often fatal human conditions. The incapacitating effects of myelindefects are typified by motor and sometimes cognitive deficiencies andare readily apparent in congenital dysmyelinating disorders as well asacquired demyelinating conditions such as multiple sclerosis andcerebral palsy. Treatment via remyelination necessitates either therestoration of the myelinating capacity of endogenous cells ortransplantation of exogenous, myelinating cells.

Transplantation of fetal human glial progenitor cells has been shown tolead to recovery in a lethally hypomyelinated mouse model (Windrem, etal., Cell Stem Cell, 2(6):553-65 (2008). While clearly establishingproof-of-principle for translation to human patients, cells from abortedhuman fetuses not only face ethical and immunological challenges;however, providing the number of cells needed on a clinical scalecurrently is not realistic. Thus, a major limiting factor to theunderstanding and treatment of myelin-related neurodegenerativedisorders is the lack of a scalable and tractable platform for the studyof oligodendrocyte development and for screening of pharmaceuticals.

Stem cell biology has garnered much attention due to the potential toimpact human health through disease modeling and cell replacementtherapy. Pluripotent stem cells in particular theoretically offer anabundant source of glial cells and their progenitors. While previousstudies have created excitement for myelin repair by clearlydemonstrating that oligodendrocytes can be derived from pluripotentcells, results have yet to yield a system to study oligodendrocytelineage that provides high cell population homogeneity without relianceon immunopanning, antibiotic resistance, or cell sorting techniques toimprove population characteristics. The excitement for myelin repair hasthus been tempered since pure populations of oligodendrocyte progenitorcells (OPCs) are difficult to obtain in clinically-relevant quantities.Methods for providing pure and plentiful glial cells are necessary toenable therapy through transplantation.

There is thus a need in the art for production of cells and cellpopulations of the glial lineages, and in particular for OPCs andoligodendrocytes. The present invention addresses this need.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present invention provides methods for generating glial cellscomprising growing mammalian pluripotent cells and/or neural precursorcells under conditions that induce differentiation of the mammalianpluripotent cells and/or neural precursor cells into cells and cellpopulations of the glial lineage. The present invention also providesmethods for the generation, expansion and use of populations ofmammalian glial cells, including populations of oligodendrocyteprogenitor cells, oligodendrocytes, and astrocytes. Moreover, theproduction of oligodendrocyte progenitor cells, oligodendrocytes, andastrocytes by the methods described herein requires far less time thanmethods used in the art currently.

Thus, the in one embodiment, the present invention provides a method forgenerating mammalian neuroectoderm comprising: providing pluripotentcells; and inducing development of the pluripotent cells intoneuroectoderm cells by culturing the pluripotent cells in the presenceof one or more inhibitors of an activin-nodal pathway and one or moreinhibitors of a bone morphogenetic protein pathway.

In another embodiment, the present invention provides a method forgenerating mammalian patterned neuroectoderm comprising: providing apluripotent cell; inducing development of the pluripotent cell intoneuroectoderm by culturing the pluripotent cell in the presence of oneor more inhibitors of an activin-nodal pathway and one or moreinhibitors of a bone morphogenetic protein pathway; and inducingdevelopment of patterned neuroectoderm by culturing the neuroectoderm inthe presence of one or more of sonic hedgehog, retinoic acid and noggin.

In yet another embodiment, the present invention provides a method forgenerating mammalian oligodendrocyte progenitor cells comprising:providing a pluripotent cell; inducing development of the pluripotentcell into neuroectoderm by culturing the pluripotent cell in thepresence of one or more inhibitors of an activin-nodal pathway and oneor more inhibitors of a bone morphogenetic protein pathway; inducingdevelopment of patterned neuroectoderm by culturing the neuroectoderm inthe presence of one or more of sonic hedgehog, retinoic acid and noggin;and inducing development of oligodendrocyte progenitor cells byculturing the patterned neuroectodermal cells in the presence of one ormore of a fibroblast growth factor, a platelet-derived growth factor andsonic hedgehog.

In some aspects of these embodiments, the pluripotent cell is amammalian epiblast stem cell, and in other aspects the pluripotent cellis a neural precursor cell. In some embodiments, the pluripotent cell isa rodent embryonic stem cell (ESC) or a rodent induced pluripotent cell(iPC), in which case the rodent ESCs or iPCs are first cultured in thepresence of a JAK/STAT inhibitor, such as JAK Inhibitor I, todifferentiate the cells into an epiblast-like cell before continuingdifferentiation to neuroectoderm.

In some aspects of these embodiments, the inhibitor of the activin-nodalpathway is SB431542, and the one or more inhibitors of the bonemorphogenetic protein pathway is selected from dorsomorphin, LDN-193189or noggin. In some aspects of these embodiments, 75% or more of cellsresulting from the culture of the pluripotent cells are neuroectodermcells, and in other aspects, 80%, 85%, 90%, 95%, 99% or more of cellsresulting from the culture of the pluripotent cells are neuroectodermcells.

In some aspects, one of retinoic acid, sonic hedgehog or noggin is usedin the culture of the neuroectoderm to produce patterned neuroectoderm,and in some aspects two of retinoic acid, sonic hedgehog or noggin areused in the culture of the neuroectoderm to produce patternedneuroectoderm. In yet other aspects all three of retinoic acid, sonichedgehog and noggin are used in the culture of the neuroectoderm toproduce patterned neuroectoderm. In some aspects, one or more of afibroblast growth factor, a platelet-derived growth factor and sonichedgehog are used to induce development of oligodendrocyte progenitorcells from the patterned neuroectoderm, and in other aspects, all threeof a fibroblast growth factor, a platelet-derived growth factor andsonic hedgehog are used to induce development of oligodendrocyteprogenitor cells from patterned neuroectoderm. In some aspects, thefibroblast growth factor is PGF2 and the platelet-derived growth factoris PDGF-AA. neuroectoderm. In some aspects of these embodiments, 75% ormore of cells resulting from the culture of the patterned neuroectodermare oligodendrocyte progenitor cells, and in other aspects, 80%, 85%,90%, 95%, 99% or more of cells resulting from the culture of thepatterned neuroectoderm are oligodendrocyte progenitor cells.

Further aspects of the invention include pharmaceutical compositionscomprising the oligodendrocyte progenitor cells of the invention,pharmaceutical compositions comprising the oligodendrocyte progenitorcells of the invention along with neurons or neuron precursor cells,diagnostic tools comprising the oligodendrocyte progenitor cells of theinvention, and research tools comprising the oligodendrocyte progenitorcells of the invention. Yet other aspects include treating a medicalcondition of the CNS in a subject comprising administering to thesubject a therapeutically effective amount of the oligodendrocyteprogenitor cells produced by methods of the invention.

Yet another embodiment of the invention provides a method for generatingmammalian oligodendrocyte progenitor cells and maintaining the mammalianoligdendrocyte progenitor cells in culture comprising: providing apluripotent cell; inducing differentiation of the pluripotent cell intoneuroectoderm by culturing the pluripotent cell in the presence of oneor more inhibitors of an activin-nodal pathway and one or moreinhibitors of a bone morphogenetic protein pathway; inducing developmentof patterned neuroectoderm by culturing the neuroectoderm in thepresence of one or more of sonic hedgehog, retinoic acid and noggin; andinducing development of oligodendrocyte progenitor cells by culturingthe patterned neuroectoderm in the presence of a fibroblast growthfactor, a platelet-derived growth factor and sonic hedgehog; andmaintaining the oligodendrocyte progenitor cells by culturing theoligodendrocyte cells in the presence of an activator of Wnt-β-cateninsignaling or in the presence of fibroblast growth factor (FGF),platelet-derived growth factor (PDGE) and sonic hedgehog (SHH).

In some aspects of this embodiment of the invention, 75% or more ofcells resulting from the culture of the patterned neuroectoderm areoligodendrocyte progenitor cells, and in other aspects, 80%, 85%, 90%,95%, 99% or more of cells resulting from the culture of the patternedneuroectoderm are oligodendrocyte progenitor cells. In some aspects, 75%or more of cells in the maintained culture of the oligodendrocyteprogenitor cells are oligodendrocyte progenitor cells, and in otheraspects, 80%, 85%, 90%, 95%, 99% or more of cells in the maintainedculture of the oligodendrocyte progenitor cells are oligodendrocyteprogenitor cells. In some aspects of this embodiment of the invention,the activator of Wnt-β-catenin signaling is a GSK3β inhibitor, and inspecific aspects, the GSK3β inhibitor is CHIR99021.

Additional aspects of this embodiment of the invention include methodsfor generating oligodendrocytes from the oligodendrocyte progenitorcells maintained in culture, comprising culturing the oligodendrocyteprogenitor cells in the absence of FGF and PDGF and in the presence of afactor to promote differentiation. In some aspects the oligodendrocytedifferentiation activator is thyroid hormone (T3). Additional aspects ofthis embodiment of the invention include methods for generatingastrocytes from the oligodendrocyte progenitor cells maintained inculture, comprising culturing the oligodendrocyte progenitor cells inthe presence of a bone morphogenetic protein and a JAK/STAT pathwayactivator.

Other embodiments and aspects of the invention are described in theDetailed Description below.

DESCRIPTION OF THE FIGURES

FIG. 1 is a general schematic showing the steps of directeddifferentiation of pluripotent cells into cells of the oligodendrocytecell fate.

FIG. 2 is a bar chart illustrating results obtained of gene expressionchanges during transition of EpiSCs to neuroectoderm.

FIG. 3 is a bar chart illustrating results obtained of gene expressionchanges during transition of patterned neuroectoderm into OPCs.

FIG. 4 is a bar chart illustrating results obtained of gene expressionchanges during transition of OPCs to oligodendrocytes.

FIG. 5 is a bar chart illustrating the suppression of expression ofmature oligodendrocyte markers upon treatment of OPCs with a GSK3βinhibitor Chir099021.

DEFINITIONS

The terms used herein are intended to have the plain and ordinarymeaning as understood by those of ordinary skill in the art. Thefollowing definitions are intended to aid the reader in understandingthe present invention, but are not intended to vary or otherwise limitthe meaning of such terms unless specifically indicated.

The terms “astrocytes” and “astroglia” refer to glial cells that anchorneurons to their blood supply. Astrocytes of the present invention referto both protoplasmic and fibrous astrocytes. Astrocytes of the presentinvention are characterized by expression of one or more of glialfibrillary acidic protein (GFAP), S100 beta, glutamine sythetase, GLASTor GLT1 and have at least one astrocytic structural or functionalphenotype. Astrocytic structural phenotypes include a round nucleus, a“star shaped” body and many long processes that end as vascular footplates on the small blood vessels of the CNS; further examples ofstructural astrocytic phenotypes may be found in the followingmaterials: Reynolds and Weiss, Science, 255:1707-1710 (1992); Reynolds,et al., J. Neurosci, 12:4565-4574 (1992); and Kandel, et al., Principlesof Neuroscience, Third Ed. (1991) (Appleton & Lange).

A “binding agent” is any molecule that binds to one or more regions onor in a cell of a particular cell fate via association by chemical orphysical means. For the purposes of the present invention, a bindingagent preferably selectively interacts with a cell surface molecule orintracellular protein or moiety that is unique to cells of a particularcell fate, e.g., oligodendrocyte progenitor cells. Examples of bindingagents that can be used in this invention include, but are notrestricted to: peptides, proteins (including derivatized or labeledproteins); antibodies or fragments thereof; small molecules; aptamers;carbohydrates and/or other non-protein binding moieties; derivatives andfragments of naturally-occurring binding partners; peptidomimetics; andpharmacophores.

The term “biological process” as used herein includes both normalphysiological processes, such as remyelination, neuroprotection, etc.,as well as pathological processes, e.g., those involved in diseases andconditions such as autoimmune diseases, neurodegenerative diseases,diseases involving genetic dysfunction, and the like.

The term “diagnostic tool” as used herein refers to any composition orassay of the invention used in order to carry out a diagnostic test orassay on a patient sample. As a diagnostic tool, the composition of theinvention may be considered a collection of analyte specific reagents,and as such may form part of a diagnostic test regulated by a federal orstate agency.

The term “excipient” refers to an inert substance added to apharmaceutical composition of the invention to further facilitateadministration of the therapeutic cells. Examples, without limitation,of excipients include saline, calcium carbonate, calcium phosphate,various sugars and types of starch, cellulose derivatives, gelatin,vegetable oils and polyethylene glycols.

The term “glial cells” and “glia” refer to non-neuronal precursor and/orfully-differentiated cells in the nervous system that provide supportand nutrition, maintain homeostasis, form myelin, and participate insignal transmission. Examples of glial cells of the present inventioninclude but are not limited to oligodendrocyte progenitor cells,oligodendrocytes and astrocytes.

The terms “introducing”, “introduction” and the like when used in thecontext of delivery of an agent to a cell (e.g., a wnt pathway activatoror a activin-nodal pathway inhibitor) refer to the delivery of the agentin any biologically effective form, including but not limited topeptides, proteins (including derivatized or labeled protein),antibodies or fragments thereof, small molecules, aptamers,peptidomimetics, and/or pharmacophores. The term “introducing” is alsointended to encompass the introduction through genetic means, e.g., theintroduction of a gene expression vector such as a viral vector (e.g.,an adenoviral vector or a lentiviral vector) or an epigenetic vector.

The term “oligodendrocyte” refers to mature well-differentiatedoligodendrocytes. Mature oligodendrocytes may be distinguished fromoligodendrocyte progenitor cells both by structural and functionalphenotypes. Examples of mature oligodendrocyte functional phenotypesinclude but are not limited to expression of one or more markers such asproteolipid protein (PLP), myelin basic protein (MBP), myelin-associatedglycoprotein (MAG), myelin oligodendrocyte glycoprotein (MOG), and/orone or more galactocerebrosides (O1, Ga1C). Examples of matureoligodendrocyte structural phenotypes include but are not limited to, abranched and ramified phenotype and the ability to effect myelination.

The terms “oligodendrocyte progenitor cells” and “OPCs” as used hereinrefer to cells that have the capacity to differentiate intooligodendrocytes. OPCs may be distinguished from oligodendrocytes bothby structural and functional phenotypes. Examples of an oligodendrocyteprogenitor cell functional phenotype include, but are not limited to,cells that are mitotic (i.e., that can divide and be expanded for threeor more passages in culture), have migratory capacity, and the potentialto differentiate into a myelinating phenotype to effect myelination invivo and in vitro.

The term “pharmaceutical composition” refers to a preparation of one ormore of the cells of the invention described herein, with at least onepharmaceutically suitable excipient.

The term “pharmaceutically acceptable carrier” refers to a carrier or adiluent that facilitates delivery and/or the biological activity andproperties of the administered cells. Examples without limitation ofcarriers are propylene glycol, saline, emulsions and mixtures of organicsolvents with water.

The term “pluripotent cells” refers to cells that are capable of bothdifferentiating into more specialized cell types (e.g., glial cells)having a particular, specialized function (i.e., “fully differentiated”cells) and the ability to give rise to cells having the same or similarundifferentiated state.

The term “research tool” as used herein refers to any cell compositionof the invention or use of the cells or cell compositions of theinvention for scientific inquiry, either academic or commercial innature, including the development of pharmaceutical and/or biologicaltherapeutics. The research tools of the invention are not intended to betherapeutic or to be subject to regulatory approval; rather, theresearch tools of the invention are intended to facilitate research andaid in such development activities, including any activities performedwith the intention to produce information to support a regulatorysubmission.

The term “selectively binds,” “selective binding” and the like as usedherein, when referring to a binding partner (e.g., protein, nucleicacid, antibody, etc.), refers to a binding reaction which isdeterminative of the presence of a composition (typically a cell marker)in heterogeneous population of molecules (e.g., proteins and otherbiologics). Thus, under designated assay conditions, the binding partnerwill bind to a composition of the invention at least two times thebackground and will not substantially bind in a significant amount toother compositions (cell markers) present in the sample. Typically,specific binding will be at least five times background signal or noiseand more typically more than 10 to 100 times background. Thus, underdesignated conditions the binding partner binds to its particular“target” composition and does not bind in a significant amount to othermolecules present in the sample.

The term “small molecule” as used herein refers to a molecule of a sizecomparable to those organic molecules generally used in chemistry-basedpharmaceuticals. The term excludes biological macromolecules (e.g.,proteins, nucleic acids, etc.). Preferred small organic molecules rangein size up to about 5000 Da, more preferably up to 2000 Da, and mostpreferably up to about 1000 Da.

As used herein, the terms “treat,” “treatment,” “treating,” and thelike, refer to obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse affect attributable to the disease. “Treatment,” as used herein,covers any treatment of a disease in a mammal, particularly in a human,and includes: (a) preventing the disease from occurring in a subjectwhich may be predisposed to the disease but has not yet been diagnosedas having it; (b) inhibiting the disease, i.e., arresting itsdevelopment; and (c) relieving the disease, e.g., causing regression ofthe disease, e.g., to completely or partially remove symptoms of thedisease.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, mammalian cell culture, biochemistry, andsequencing technology, which are within the skill of those who practicein the art. Such conventional techniques include polymer arraysynthesis, hybridization and ligation of polynucleotides, and detectionof hybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the examples herein. However,other equivalent conventional procedures can, of course, also be used.Such conventional techniques and descriptions can be found in standardlaboratory manuals such as Butler (2004), Animal Cell Culture (BIOSScientific); Picot (2005), Human Cell Culture Protocols (Humana Press),Davis (2002), Basic Cell Culture, Second Ed. (Oxford Press); Lanza, etal., (Eds.) (2009), Essentials of Stem Cell Biology, Second Ed.(Elsevier Academic Press); Lanza, (Ed.) (2009), Essential Stem CellMethods (Elsevier Academic Press); Loring, et al. (Eds.) (2007), HumanStem Cell Manual (Elsevier Academic Press); Freshney (2010), Culture ofAnimal Cells (John Wiley & Sons); Ozturk and Hu (2006), Cell CultureTechnology for Phamaceutical and Cell-Based Therapies (CRC Press);Sambrook and Russell (2006), Condensed Protocols from Molecular Cloning:A Laboratory Manual; and Sambrook and Russell (2002), Molecular Cloning:A Laboratory Manual (both from Cold Spring Harbor Laboratory Press);Stryer, L. (1995) Biochemistry, Fourth Ed. (W.H. Freeman); Nelson andCox (2000), Lehninger, Principles of Biochemistry, Third Ed. (W. H.Freeman); and Berg et al. (2002) Biochemistry, Fifth Ed. (W. H.Freeman); all of which are herein incorporated in their entirety byreference for all purposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “an oligodendrocyteprogenitor cell” refers to one or more cells of glial cell fate, andreference to “sorting” or “inducing” includes reference to equivalentsteps and methods known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing devices, formulations and methodologies that may be used inconnection with the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the invention subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, features and procedures well known to thoseskilled in the art have not been described in order to avoid obscuringthe invention.

Culture Methods of the Invention to Differentiate and Create CellPopulations

The present invention is based on the novel discovery that pluripotentstem cells can be differentiated through a defined series ofdevelopmental transitions into cells and cell populations of the gliallineage, recapitulating the complex signaling environment present in thedeveloping embryo. This includes differentiating pluripotent stem cellsinto relatively pure populations of expandable cells including OPCs,oligodendrocytes and astrocytes. The methods provide the ability tocreate large quantities of cells of the glial lineage, includingoligodendrocyte progenitor cells from pluripotent stem cells. Such OPCscan then be either expanded into additional OPCs or furtherdifferentiated into mature cells such as oligodendrocytes andastrocytes. These glial cells can be differentiated directly frompluripotent cells, or they may be generated from existing neuralprecursor cells that are already partially driven down the ectodermalcell fate pathway.

FIG. 1 is a general schematic showing the steps of a method 100 fordirected differentiation of pluripotent cells into expandablepopulations of OPCs, myelinating oligodendrocytes, and astrocytes. At101, pluripotent cells are provided. Numerous types of pluripotent cellscan be used in the methods of the present invention. The pluripotentcells are preferably mammalian stem cells, although in certain aspectsavian pluripotent cells may be used in the generation of the cells andcell populations of the invention.

In some aspects of the invention, the pluripotent cells are rodentpluripotent cells. Such rodent pluripotent cells include rat or mouseinduced pluripotent cells, rat or mouse embryonic stem cells (mEScells), such as those described in or derived as described in, e.g.,U.S. Pat. No. 6,190,910; rat or mouse epiblast-derived stem cells(EpiSCs), such as those described in USSN 2010/0064380 and Tesar, etal., Nature, 448(7150):196-9 (2007), Epub Jun. 27, 2007, both of whichare incorporated herein by reference; rat or mouse induced epiblast stemcells, rodent embryonic germ cells (EGCs) that have been derived fromprimordial germ cells (PGCs) of the midgestation embryo (Matsui, et al.,Cell, 70(5):841-7 (1992); Resnick, et al., Nature, 359(6395):550-1(1992); and multipotent germline stem cells (mGSCs) that have beengenerated from explanted neonatal testicular cells (Kanatsu-Shinohar,aet al., Cell, 119(7):1001-12 (2004)), adult testicular cells (Guan, etal., Nature, 440(7088):1199-203 (2006), Epub Mar 24, 2006; Seandel, etal., Nature,;449(7160):346-50 (2007); Ko, et al., Cell Stem Cell,5(1):87-96 (2009)), or mouse testicular cells. In the case of rodentembryonic stem cells or rodent induced pluripotent cells, the cells arefirst differentiated into an epiblast state using, e.g., growth in aculture medium containing a JAK/STAT inhibitor such as JAK Inhibitor Ifor two to four days.

In certain aspects, the pluripotent cells are derived from rodentstrains with clinically relevant genotypes, e.g., mice having specificmutations or polymorphisms associated with clinical sensitivities orpathologies in humans. Mouse embryonic stem cells having variousmutations or polymorphisms allow the production of glial cells withthese mutations, which are useful in studying disease progression andproducing cell populations, e.g., for identifying therapeutic candidateagents or toxicity testing of candidate or existing therapeutic agents.

In other aspects, other mammalian pluripotent cells may be utilized,depending on e.g., the mammal to be treated or the animal model ofinterest. Such mammals include cats, dogs, horses, pigs, cows, sheep,etc., and primates.

In other aspects, the pluripotent cells used in the methods of theinvention are human cells. For example, human embryonic stem cells canbe isolated from human blastocysts or derived from a delayed blastocyststage (e.g., those described in WO2006/040763). Pluripotent cell lineshave also been derived from other embryonic and adult tissues uponexplantation in culture, and techniques are known in the art forpreparing such human induced or derived pluripotent cells. Common to allof these cell types is their origin from either early embryos or germlineage cells, which appear to be the only cells that harbor anepigenetic conformation that is permissive for spontaneous conversioninto a pluripotent state. A molecular commonality among these cell typesis the expression of endogenous Oct4, which may thus serve as a valuablepredictive marker for whether or not a cell can give rise to pluripotentcell lines useful to generate the OPCs, OPC populations,oligodendrocytes and astrocytes of the invention. In some cases of humanembryonic stem cells or human induced pluripotent cells, the cells arefirst differentiated into an epiblast state using, e.g., growth in aculture medium containing a JAK/STAT inhibitor such as JAK Inhibitor Ifor two to four days.

Stem cells for use in the present invention also may be derived fromhuman embryonic germ (EG) cells. For additional details on methods ofpreparation human embryonic germ cells see Shamblott et al., PNAS USA,95: 13726 (1998) and U.S. Pat. No. 6,090,622. In certain aspects, thepluripotent cells used for differentiation of the cells and cellpopulations of the glial cell lineage are induced pluripotent stemcells, such as those as described in Tokuzawa et al., Cell,113(5):631-42 (2003).

At step 102, one or more activin-nodal pathway inhibitors and one ormore bone morphogenetic protein pathway inhibitors are added to theculture medium of the pluripotent cells 101. Cell culture techniques arewell known in the art, with reference to such text books as Butler(2004), Animal Cell Culture (BIOS Scientific); Picot (2005), Human CellCulture Protocols (Humana Press); Davis (2002), Basic Cell Culture,Second Ed. (Oxford Press); Freshney (2010), Culture of Animal Cells(John Wiley & Sons); and Ozturk and Hu (2006), Cell Culture Technologyfor Phamaceutical and Cell-Based Therapies (CRC Press). For example, thepluripotent cells may be epiblast stem cells, where the medium forculture comprises Knockout DMEM supplemented with 20% Knockout SerumReplacement, Glutamax, nonessential amino acids and 0.1 mM2-mercaptoethanol (Sigma-Aldrich, St. Louis, Mo.), or other media knownin the art.

The one or more activin-nodal pathway inhibitors and one or more bonemorphogenetic protein pathway inhibitor are added to (are “introducedto”) this culture medium. Nodal is a protein that in humans is encodedby the NODAL gene, and is a member of the Transforming Growth Factorbeta superfamily. Like many other members of the TGF beta superfamily,Nodal is involved in cell differentiation. Studies of the mousecounterpart of NODAL suggest that this gene may be essential formesoderm formation and subsequent organization of left-right axialstructures in early embryonic development. A Nodal knockout in micecauses precocious differentiation to neuroectoderm and an absence of theprimitive streak and failure in formation of mesoderm, leading todevelopmental arrest just after gastrulation. Activin-nodal signalingcontributes to maintaining pluripotency of human embryonic stem cells(hESCs), which are derivatives of the inner cell mass but sharemolecular properties with epiblast tissue. Inhibition of activin-nodalsignaling results in the loss of hESC pluripotency. Examples ofinhibitors of the activin-nodal pathway include but are not limited toSB431542, SB505124, A83-01, and follistatin.

Bone Morphogenetic Proteins (BMPs) are a family of ligands that alsobelong to the TGF beta superfamily. BMPs interact with specificreceptors on a cell surface, referred to as bone morphogenetic proteinreceptors. Signal transduction through BMPRs results in phosphorylationof downstream targets. The best characterized signaling cascade is theSmad pathway, shown to be important in the development of the heart,central nervous system, and cartilage, as well as post-natal bonedevelopment. Mutations in BMPs and their antagonists, for exampleSclerostin, are associated with a number of human disorders that affectthe skeleton and other tissues. Examples of bone morphogenetic proteinpathway inhibitors include but are not limited to Dorsomorphin,LDN-193189, noggin, ALK3-Fc, ALK6-Fc, Gremlin, Chordin, and Cerberus.

Treatment of pluripotent cells, such as pluripotent epiblast stem cell,with an activin-nodal pathway inhibitor such as SB431542, an inhibitorof TGF beta superfamily Type I Activin receptor-like kinase (ALK)receptors ALK4, ALK5, and ALK7 (see Inman, et al., MolecularPharmacology, 62(1):65-74 (2002)) and a bone morphogenetic proteinpathway inhibitor such as Dorsomorphin, a selective small moleculeinhibitor of BMP signaling (see, Yu, et al., Nat Chem Biol, 4: 33-41(2008)) results in region-specific neuroectodermal cell differentiation103. The resulting neuroectoderm can then be further differentiated intopatterned neuroectoderm 105 by culture in the presence of one or more ofsonic hedgehog (SHH), retinoic acid (RA) and noggin at step 104, whichresults in the up-regulation of region-specific transcription factorsOlig2 and Nkx2.2, which are normally expressed in the ventral region ofthe developing neural tube. Differentiation to oligodendrocyteprogenitor cells is achieved by culturing the neuroepithelial cells inthe presence of a platelet-derived growth factor (PDGF), a fibroblastgrowth factor (FGF) and sonic hedgehog (SHH) at 106

Sonic hedgehog homolog (SHH) is one of three proteins in the mammaliansignaling pathway family called hedgehog, the others being deserthedgehog and Indian hedgehog. SHH is the best studied ligand of thehedgehog signaling pathway, playing a key role in regulating vertebrateorganogenesis, such as in the growth of digits on limbs and organizationof the brain. Sonic hedgehog is one of the best established example of amorphogen—a molecule that diffuses to form a concentration gradienthaving different effects on the cells of the developing embryo dependingon its concentration. Sonic hedgehog assumes various roles in patterningthe central nervous system (CNS) during vertebrate development, andremains important in the adult, controlling cell division of adult stemcells. More recently, sonic hedgehog has also been shown to act as anaxonal guidance cue

Retinoic acid (RA) is a metabolite of vitamin A (retinol) that mediatesthe functions of vitamin A required for growth and development. Retinoicacid is required in chordate animals which includes all higher animalsfrom fishes to humans. During early embryonic development, retinoic acidgenerated in a specific region of the embryo helps determine positionalong the embryonic anterior/posterior axis by serving as anintercellular signaling molecule that guides development of theposterior portion of the embryo. It acts through Hox genes, whichultimately control anterior/posterior patterning in early developmentalstages.

Noggin, also known as NOG, is a protein which in humans is encoded bythe NOG gene. Noggin inhibits TGF-β signal transduction by binding toTGF-β family ligands and preventing them from binding to theircorresponding receptors.

Noggin plays a key role in neural induction by inhibiting BMP4, alongwith other TGF-β signaling inhibitors such as chordin and follistatin.Mouse knockout experiments have demonstrated that noggin also plays acrucial role in bone development, joint formation, and neural tubefusion. The secreted polypeptide noggin, encoded by the NOG gene, bindsand inactivates members of the transforming growth factor-betasuperfamily signaling proteins, such as bone morphogenetic protein-4.Noggin appears to have pleiotropic effect, both early in development aswell as in later stages.

Platelet-derived growth factor (PDGF) is one of numerous growth factorsthat regulate cell growth and division, playing a significant role inangiogenesis, embryonic development, cell proliferation, and cellmigration. In chemical terms, platelet-derived growth factor is dimericglycoprotein composed of two A (-AA) or two B (-BB) chains or acombination of the two (-AB). PDGFs are mitogenic during earlydevelopmental stages, driving the proliferation of undifferentiatedmesenchyme and some progenitor populations. During later maturationstages, PDGF signaling has been implicated in tissue remodeling andcellular differentiation, and in inductive events involved in patterningand morphogenesis. In addition to driving mesenchymal proliferation,PDGFs have been shown to direct the migration, differentiation andfunction of a variety of specialized mesenchymal and migratory celltypes, both during development and in the adult animal. Other growthfactors in this family include vascular endothelial growth factors B andC, and placenta growth factor (P1GF).

Fibroblast growth factors, or FGFs, are a family of growth factorsinvolved in angiogenesis, wound healing, and embryonic development. FGFsare multifunctional proteins with a wide variety of effects; they aremost commonly mitogens but also have regulatory, morphological, andendocrine effects. They have been alternately referred to as“pluripotent” growth factors and as promiscuous growth factors due totheir multiple actions on multiple cell types. In the case of FGF, fourreceptor subtypes can be activated by more than twenty different FGFligands. The functions of FGFs in developmental processes includemesoderm induction, antero-posterior patterning, limb development,neural induction and neural development, and in mature tissues/systemsangiogenesis, keratinocyte organization, and wound healing processes.

The oligodendrocyte progenitor cells 107 formed from the patternedneuroectoderm in method 100 can be maintained in culture withoutdifferentiation through the introduction of a Wnt pathway activator,preferably using one or more GSK3β inhibitors, or by culture in thepresence of fibroblast growth factor (FGF), platelet-derived growthfactor (PDGF) and sonic hedgehog (SHH) at 108. Exemplary GSK3βinhibitors that may be used in the methods of the invention include, butare not limited to, lithium chloride (LiCl), 6-bromoindirubin-3′-oxime(BIO), Chiron 99021 and those compounds described in U.S. Pat. No.7,300,943 to Gabriel, et al., which is incorporated herein by referencein its entirety. The Wnt signaling pathway is a network of proteins bestknown for their roles in embryogenesis and cancer, but also involved innormal physiological processes in adult animals. Wnt proteins activatevarious pathways in the cell that can be categorized into the canonicaland noncanonical Wnt pathways. The canonical Wnt pathway describes aseries of events that occur when Wnt proteins bind to cell-surfacereceptors of the Frizzled family, causing the receptors to activateDisheveled family proteins, ultimately resulting in a change in theamount of β-catenin that reaches the nucleus. Disheveled is a keycomponent of a membrane-associated Wnt receptor complex, which, whenactivated by Wnt binding, inhibits a second complex of proteins thatincludes axin, GSK3, and the protein APC. The axin/GSK-3/APC complexnormally promotes the proteolytic degradation of the β-cateninintracellular signaling molecule. After this β-catenin destructioncomplex is inhibited, a pool of cytoplasmic β-catenin stabilizes, andsome β-catenin is able to enter the nucleus and interact with TCF/LEFfamily transcription factors to promote specific gene expression.Several protein kinases and protein phosphatases have been associatedwith the ability of the cell surface Wnt-activated Wnt receptor complexto bind axin and disassemble the axin/GSK3 complex. Phosphorylation ofthe cytoplasmic domain of LRP by CK1 and GSK3 can regulate axin bindingto LRP. The protein kinase activity of GSK3 appears to be important forboth the formation of the membrane-associated Wnt/FRZ/LRP/DSH/Axincomplex and the function of the Axin/APC/GSK3β-catenin complex.Phosphorylation of β-catenin by GSK3 leads to the destruction ofβ-catenin.

As an alternative to maintaining the OPCs in a progenitor cell state,the OPCs can be further differentiated into cells of the glial lineagethrough the control of other molecular mechanisms. In some embodiments,the OPCs generated using the methods of the invention are differentiatedinto myelinating oligodendrocytes 111 in vitro by withdrawal of FGF andPDGF and addition of T3 110. T3, triiodothyronine, is a thyroid hormoneand affects almost every physiological process in the body, includinggrowth and development, metabolism, body temperature, and heart rate.Production of T3 and its prohormone thyroxine (T4) is activated bythyroid-stimulating hormone (TSH), which is released from the pituitarygland. This pathway is regulated via a closed-loop feedback process.Elevated concentrations of T3, and T4 in the blood plasma inhibit theproduction of TSH in the pituitary gland; as concentrations of thesehormones decrease, the pituitary gland increases production of TSH, andby these processes, a feedback control system is set up to regulate theamount of thyroid hormones that are in the bloodstream. T3 has profoundeffect upon the developing embryo and infants, affecting the lungs andinfluences the postnatal growth of the central nervous system. T3 alsostimulates the production of myelin, the production ofneurotransmitters, and the growth of axons, and is also important in thelinear growth of bones.

In yet another alternative, the OPCs generated using the methods of theinvention are differentiated into astrocytes 113 in vitro using a bonemorphogenetic protein (BMP) and a JAK/STAT pathway activator 112. Asdescribed above, BMPs are a family of ligands that belong to the TGFbeta superfamily, originally recognized for their ability to induceendochondral bone formation. One BMP particularly suited for use in theinvention for differentiation of OPCs into astrocytes is BMP4. In humanembryonic development, BMP4 is a signaling molecule required for theearly differentiation of the embryo and establishment of adorsal-ventral axis. BMP4 is secreted from the dorsal portion of thenotochord, and acts in concert with SHH to establish a dorsal-ventralaxis for the differentiation of later structures. In addition, BMP4stimulates differentiation of overlying ectodermal tissue.

The janus kinase (JAK)-signal transducer and activator of transcription(STAT) pathway plays a critical role in the signaling of a wide array ofcytokines and growth factors leading to various cellular functions,including proliferation, growth, hematopoiesis, and immune response. Thebinding of cytokines and growth factors to their corresponding receptorsactivates JAK, which then phosphorylates the receptor and STAT proteinson specific tyrosine residues. STATs then dimerize, translocate to thenucleus, bind to a consensus DNA sequence and initiate the transcriptionof target genes. Four JAK family kinases and seven STAT family membershave been identified, some being ubiquitously expressed. Amino acidsequence diversity and tissue-specific distributions account for thediverse roles of STATs in response to extracellular cytokines Activatorsof JAK/STAT pathways include leukemia inhibitory factor (LIF), IL-6,cntf, CT-1, and OSM.

Using the methods of the invention outlined above, pluripotent cells canthus be differentiated into pure populations of clinically-relevant,OPCs that provide a tractable platform for defining the molecularregulation of oligodendrocyte development, exploring the causative celland molecular defects that function in congenital disorders impactingthe oligodendrocyte lineage, and for high-throughput drug screening.Moreover, the cell populations of the invention may be used incell-based therapies to restore functional myelination. Alternatively,the OPCs can be further differentiated into myelinating oligdendrocytesand astrocytes that may be used in vitro or in vivo for drug screeningor for cell-based therapies.

Cell Transplantation

Numerous methods for introducing the cells and cell populations of theinvention into a subject may be used. An important aspect of theinvention relates to a method of treating a subject having a conditionmediated by a loss of myelin and/or an inability of oligodendrocytes toremyelinate nerve fibers by administering to the subject oligodendrocyteprogenitor cells under conditions effective to treat the condition. Forinjection, cells of the pharmaceutical composition may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hank's solution, Ringer's solution, or physiological salt buffer.

Administration of the pharmaceutical cell composition by injectionincludes intraparenchymal injections into the affected portion of thebrain itself as well as introduction of the cells at a more distal sitein the brain, brain stem or CNS, with the cells then migrating to theaffected part of the brain. Administration typically involves cell dosesranging from 1×10⁴ to 1×10⁹, depending on the extent of desiredremyelination. Dosage amount and interval may be adjusted individuallyto levels that are sufficient to effectively regulate remyelination bythe implanted cells. Dosages necessary to achieve the desired effectwill depend on individual characteristics and the route(s) ofadministration.

Depending on the severity and responsiveness of the condition to betreated, dosing may comprise a single administration or a plurality ofadministrations, with course of treatment lasting from several days toseveral weeks or until diminution of the disease state is achieved. Theamount of the pharmaceutical cell composition to be administered will,of course, be dependent on the individual being treated, the severity ofthe affliction, the manner of administration, the judgment of theprescribing physician, etc. The dosage and timing of administrationoptimally will be responsive to a careful and continuous monitoring ofthe subject's changing condition. For example, a treated MultipleSclerosis patient will be administered an amount of cells that issufficient to alleviate the symptoms of the disease, based on themonitoring indications.

The cells of the present invention may be co-administered in a“cocktail” with therapeutic agents useful in treating neurodegenerativedisorders, such as gangliosides; antibiotics, neurotransmitters,neurohormones, toxins, neurite promoting molecules; and antimetabolitesand precursors of neurotransmitter molecules. Additionally, the cells ofthe invention may be co-administered with other cells.

Following transplantation, the cells of the invention preferably survivein the diseased area for a period of time (e.g., at least six months),such that a therapeutic effect is observed. In one aspect of the presentinvention, oligodendrocyte progenitor cells are administered to thesubject after administration of radiation, e.g., to treat primary andmetastatic tumors of the central nervous system.

In certain circumstances, including those in which the OPC oroligodendrocyte deficiency is coupled with a loss of neurons, it may bedesirable to transplant mixed cell populations, such as mixtures of theOPC cell populations of the invention and neurons or neuronalprecursors. The differentiated cells of the invention can thus beco-introduced with neurons or neuronal precursors, such as thoseproduced as described in USSN 2010/0021437, which is incorporated hereinby reference. In one aspect of the invention, a subject will be treatedwith both OPC or oligodendrocyte cell populations and neurons orneuronal precursor cells.

Generally, any method known in the art can be used to monitor success oftransplantation, including both clinical and phenotypic indicators. Forexample, MRI can be used for visualizing brain white matter and studyingthe burden of demyelinating lesions as currently practiced formonitoring MS patients. Magnetic resonance spectroscopy measurement ofN-acetyl-aspartate levels can be used to assess impact on localneuron/axon survival by using paramagnetic particles to label cellsbefore transplantation, enabling cell dispersion to be tracked by MRI.Alternatively or in addition, magnetization transfer contrast can beused to monitor remyelination (Deloire-Grassin, J. Neurol. Sci.,178:10-16 (2000)). Serial neurophysiology monitoring techniques can alsobe used to assess improvement over time.

Additionally, electrophysiological measures of sensory and motor nerveconductivity, for example H-wave response, are classical methods usedfor monitoring neuropathies linked to demyelinating peripheral lesions(Lazzarini et al, Eds (2004) Myelin Biology and Disorders (ElsevierAcademic Press)).

Other approaches to more generalized phenotypic neurophysiologicalassessment are described in Leocani et al., Neurol Sci., 21(4 Suppl2):S889-91 (2000), which may be useful for interventions aimed atmultifocal or more diffuse myelin repair. For example, demyelinationcauses alterations of stature (trembling, shivering) and locomotion, andchildren with leukodystrophies have motor and intellectual retardation.Improvement in these states may be assessed to monitor therapeuticsuccess.

Applicable Disease States

The cell populations created using the methods of the invention can beused for research of demyelinating states, including the identificationand development of drugs and therapeutic interventions in multipledisease states involving glial cells, and in particular diseasesinvolving the CNS. Exemplary diseases that may be studied and for whichtherapeutic interventions may be identified using the cell populationsof the invention are described in more detail below.

Multiple sclerosis (MS), a progressive, neurodegenerative disease of the

CNS, occurs most often in a relapsing/remitting form, in which a periodof demyelination is followed by a period of functional recovery (Weiner,Ann Neurol, 65:239-248 (2009)). The recovery stage involvesremyelination via migration and maturation of OPCs (Chari, Int RevNeurobiol, 79:589-620 (2007)). However, as the disease progresses,remyelination fails with progressive loss of function (Blakemore andKeirstead, J Neuroimmunol, 98:69-76 (1999)). Possible explanations forremyelination failure of intact axons include defects in OPC recruitmentto the site of demyelination or defects in OPC differentiation intomyelinating oligodendrocytes. Although studies indicate that bothaspects of OPC biology are altered in MS, the molecular mechanisms thatorchestrate these processes within the adult CNS are incompletelyunderstood.

Other conditions mediated by a loss of myelin include an ischemicdemyelination condition, an inflammatory demyelination condition, apediatric leukodystrophy, mucopolysaccharidosis, perinatal germinalmatrix hemorrhage, cerebral palsy, periventricular leukoinalacia,radiation-induced conditions, and subcortical leukoencephalopathy due tovarious etiologies, and mental illnesses, such as schizophrenia.Ischemic demyelination conditions include cortical stroke, Lacunarinfarct, post-hypoxic leukoencephalopathy, diabetic leukoencephalopathy,and hypertensive leukoencephalopathy. Inflammatory demyelinationconditions include multiple sclerosis, Schilder's Disease, transversemyelitis, optic neuritis, post-vaccination encephalomyelitis, andpost-infectious encephalomyelitis. Pediatric leukodystrophy conditionsinclude lysosomal storage diseases (e.g., Tay-Sachs Disease), Cavavan'sDisease, Pelizaeus-Merzbacher Disease, and Crabbe's Globoid bodyleukodystrophy. An example of mucopolysaccharidosis is Sly's Disease.Radiation-induced conditions include radiation-inducedleukoencephalopathy and radiation induced myelitis. Etiologies causingsubcortical leukoencephalopathy include HIV/AIDS, head trauma, andmulti-infarct states.

According to some features of the invention, the pharmaceutical cellcompositions used in therapy may comprise oligodendrocytes and themedical condition is associated with insufficient myelination. Accordingto still further features, the cells comprise astrocytes and the medicalcondition is selected from the group consisting of Alexander disease,epilepsy, Alzheimer's disease, spinal cord injury, traumatic braininjury and neurogenesis deficiencies. The subjects treated withpharmaceutical compositions comprises oligodendrocyte progenitor cellsin accordance with the present invention are preferably mammals, morepreferably humans and, most preferably, an adult or post-natal human.

Cell Populations as Research Tools for Drug Discovery and for ToxicityTesting

One significant use of the glial cell populations of the invention is asa research tool specifically for the discovery and development oftherapeutic products for modulation of one or more biological processesinvolved in diseases, disorders and/or physiological processes such asneuronal repair. The cell-based research tools may be useful in variousaspects of drug discovery and investigation, including withoutlimitation initial identification of a drug candidate, confirmation ofactivity of a drug candidate, identification of activity for an existingpharmaceutical product, and/or toxicity of a drug or drug candidate.Another use of the cell-based compositions is as a research toolspecifically used as a diagnostic tool to detect the presence or absenceof molecules necessary for the modulation of a biological processinvolved in a disease or disorder. Thus, in one aspect, the inventionincludes research tools comprising the cell compositions of theinvention, and uses of such research tools in the identification,investigation and/or confirmation of activity of selective bindingagents that are useful as therapeutic agents. The present inventionadditionally encompasses binding agents that are isolated using themethods of the invention and uses of such binding agents in either atherapeutic or a diagnostic setting.

Thus, according to yet another aspect of the present invention there isprovided a method of determining an effect of a treatment on CNSfunctionality, the method comprising subjecting cells of the presentinvention to a treatment or binding agent (e.g., drug, condition such aselectrical treatment and an irradiation treatment); and determining atleast one of a structural or functional phenotype of the treated cell ascompared to an untreated cell, thereby determining an effect of thetreatment on CNS functionality.

Determining the effect of a treatment of interest on the cells of thepresent invention can be used to identify and optimize treatmentscapable of restoring neural function via activity of glial cells, andhence can be used to identify and optimize drugs suitable for treatingneural disorders. Determining the effect of a treatment directed todiseases of the CNS or any other tissue requiring neural functionalitycan be used to assess the toxicity of such clinical treatments on CNSfunction. Thus, this aspect of the invention can be utilized todetermine the therapeutic and toxic effects of various treatments, suchas drug treatments, on neural function via assessment of the activity ofglial cells. Other aspects of the invention encompass using the cells orcell populations to obtain gene expression profiles and other changesbefore and after the cells or cell populations are subjected to atreatment.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific embodiments withoutdeparting from the spirit or scope of the invention as broadlydescribed. The present embodiments are, therefore, to be considered inall respects as illustrative and not restrictive.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperature, etc.) but some experimental errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, molecular weight is weight average molecularweight, temperature is in degrees centigrade, and pressure is at or nearatmospheric.

In general, the methods described herein and in the specific examplesbelow are applicable to mammalian pluripotent cells; however, certaincells such as rodent embryonic stem cells and induced pluripotent stemcells, and various other mammalian stem cells (including human stemcells) may require a first step to differentiate these cells intoepiblast or epiblast-like cells. In addition, the timing required forthe various differentiation steps described herein may vary betweenmammals. For example, differentiation from pluripotent cells toneuroectoderm in mice may take from 2 to 5 days using the methodsherein, where such differentiation in human pluripotent cells may takefrom 4 to 10 days. Similarly, differentiation from neuroectoderm topatterned neuroectoderm in mice may take from 1 to 2 days using themethods herein, where such differentiation in human neuroectoderm maytake from 2 to 5 days.

Example 1 Differentiation of Pluripotent Cells to Neuroectoderm

All cells were cultured at 37° C. and 5% CO₂ unless otherwise noted.Individual EpiSC lines were isolated from mice of strains 129SvEv(EpiSC5 and EpiSC7 lines), (12901 line) and 129SvEv x ICR (EpiSC9 line),and maintained in vitro in EpiSC base medium supplemented with 10 ngml⁻¹ PGF2 (R&D Systems, Minneapolis, Minn., 233-FB). Differentiation ofEpiSCs to patterned neuroectoderm was a 5-day procedure with completemedium changes every day.

EpiSC base medium consisted of Knockout DMEM (Invitrogen, Carlsbad,Calif.) supplemented with 20% Knockout Serum Replacement (KSR;Invitrogen, Carlsbad, Calif.), 2 mM Glutamax (Invitrogen, Carlsbad,Calif.), 1×nonessential amino acids (Invitrogen, Carlsbad, Calif.) and0.1 mM 2-mercaptoethanol (Sigma-Aldrich, St. Louise, Mo.). Neural basemedium consisted of DMEM/F12 (Invitrogen, Carlsbad, Calif.; 11320)supplemented with 1×N2 (R&D Systems, Minneapolis, Minn.), 1×B-27 withoutvitamin A (Invitrogen, Carlsbad, Calif.) and 2 mM Glutamax. On day 0,EpiSCs were plated under standard passaging conditions in EpiSC basemedium supplemented with 100 ng ml⁻¹ noggin (R&D Systems, Minneapolis,Minn.), 20 μM SB431542 (Sigma-Aldrich, St. Louis, Mo.; maintained as a20 mM stock solution in DMSO) and 2 μM dorsomorphin (EMD; supplied as a10 mM stock solution in DMSO). 0.1 μM LDN-193189 (Stemgent, San Diego,Calif.; maintained as a 1 mM stock solution according to themanufacturer's instructions) was used in place of dorsomorphin for someexperiments. On day 1, cultures were fed with a 1:1 mixture of EpiSCbase medium and neural base medium supplemented with 100 ng ml⁻¹ noggin,20 μM SB431542 and 2 μM dorsomorphin On day 2, cultures were fed withneural base medium supplemented with 100 ng ml⁻¹ noggin, 20 μM SB431542and 2 μM dorsomorphin On day 3, cultures were fed with neural basemedium supplemented with 100 ng ml⁻¹ noggin. On day 4, cultures were fedwith neural base medium supplemented with 100 ng ml⁻¹ noggin, 10 μMretinoic acid (Sigma-Aldrich, St. Louis, Mo.; maintained as a 20 mMstock solution in DMSO) and 200 ng ml⁻¹ SHH (R&D Systems; C24II).

Example 2 Differentiation of EpiSC-derived, patterned neuroectoderm toOPCs.

Day-5 patterned neuroectodermal cells were released from the culturedish using 1.5 mg ml⁻¹ collagenase IV (Invitrogen, Carlsbad, Calif.) anddissociated to a single cell suspension using TrypLE Select (Invitrogen,Carlsbad, Calif.). Cells were counted and plated at 4×104 cells cm−2 onNunclon-Δ plates coated with 0.1 mg ml⁻¹ poly(L-ornithine)(Sigma-Aldrich, St. Louis, Mo.) followed by 10 μg ml⁻¹ laminin (Sigma;L2020). Cells were grown in OPC medium consisting of neural base mediumsupplemented with 20 ng ml⁻¹ FGF2, 20 ng ml⁻¹ PDGF-AA (R&D Systems,Minneapolis, Mo.) and 200 ng ml⁻¹ SHH, and fed every other day for 5days. At this point cultures consisted of a highly pure population ofproliferating OPCs.

Example 3 OPC Culture

Cultures of EpiSC-derived OPCs were maintained and expanded in neuralbase medium supplemented with 20 ng ml⁻¹ FGF2, 20 ng ml⁻¹ PDGF-AA and200 ng ml⁻¹ SHH or 20 ng ml⁻¹ PDGF-AA, 200 ng ml⁻¹ SHH, 100 ng ml⁻¹noggin, 10 μM dibutyryl cyclic-AMP sodium salt (Sigma-Aldrich, St.Louis, Mo.), 100 ng ml⁻¹ IGF-1 (R&D Systems, Minneapolis, Mo.), and 10ng ml⁻¹ NT3 (R&D Systems, Minneapolis, Mo.) and fed every other day.OPCs were grown on Nunclon-Δ plates coated with poly(L-ornithine) andlaminin Cells were passaged every 3-5 days with TrypLE Select andtypically seeded at 2×10⁴ cells cm⁻². OPCs could be readily frozen orthawed and were cryopreserved in DMEM supplemented with 10% FBS(Invitrogen, Carlsbad, Calif.) and 10% DMSO (Sigma-Aldrich, St. Louis,Mo.). For the cumulative OPC experiment, at ‘passage 0’ 4×10⁴ cells cm⁻²were seeded. Subsequently, cells were passaged at 80-90% confluence andseeded at 2×10⁴ cells cm ². Total cell number at each passage wascounted with a hemocytometer. Rates of growth were determined at eachpassage and extended to the entire population of cells to yield acumulative count.

Example 4 Differentiation of EpiSC-Derived OPCs

For differentiation of OPCs into oligodendrocytes, cells were plated at2.2×10⁴ cells cm⁻² and induced with neural base medium supplemented with0.4 ng ml⁻¹ T3 (Sigma-Aldrich, St. Louis, Mo.), 200 ng ml⁻¹ SHH, 100 ngml⁻¹ noggin, 10 μM dibutyryl cyclic-AMP sodium salt, 100 ng ml⁻¹ IGF-1and 10 ng ml⁻¹ NT3. Attempts to modulate the outcome of thisdifferentiation paradigm included treatments with Jagl (R&D Systems,Minneapolis, Minn.), CHIR99021 (Stemgent, San Diego, Calif.), LIF(Millipore, Billerica, Mass.) and/or BMP4 (R&D Systems, Minneapolis,Minn.).

Example 4 Time-Lapse Imaging of EpiSC-Derived OPC Differentiation andImmunostaining

For live-cell imaging experiments, EpiSC-derived OPCs were plated ontopoly(1-ornithine) and laminin-coated glass-bottom microwell dishes(MatTek, Ashland, Mass., P35G-1.5-14-C). Cells were seeded at a densityof 3.1×10⁴ cells cm⁻² in differentiation medium (T3, SHH, noggin, cAMP,IGF1 and NT3). After allowing the cells to adhere for 3 hours, imageswere collected every 10 minutes for up to 72 hours with a Hamamatsu Orcacharge-coupled device (CCD) camera on an inverted microscope (LeicaDMI6000B) outfitted with a precision scanning stage (Marzhauser,Wetslar, Germany) in a live-cell incubation chamber and cover (Pecon,Erbach, Germany) at 37° C. and 5% CO₂. Adaptive focus control was usedto maintain multiposition focus stability over time. Movies weregenerated using Image Pro Plus and Adobe Premiere Pro CS5.

In vitro-cultured cells were prepared for immunostaining by fixation in4% (vol/vol) paraformaldehyde (Electron Microscopy Sciences, Hatfield,Pa.) for 15 minutes and subsequent permeabilization for 10 minutes with0.2% Triton X in PBS (pH 7.4). Cells were then blocked for nonspecificbinding with 10% normal goat serum (Abcam) or 10% normal donkey serum(Abcam, Cambridge, Mass.) in PBS for 1-2 hours at room temperature(21-24° C.). Primary antibodies were diluted in blocking solution andincubated with the samples either overnight at 4° C. or for 1 hour atroom temperature. Samples were rinsed with PBS and incubated with theappropriate Alexa Fluor-labeled secondary antibodies (Invitrogen; 4 μgml⁻¹) for 1 hour at room temperature. For visualization of nuclei,samples were incubated with 1 μg ml⁻¹ DAPI (Sigma-Aldrich, St. Louis,Mo.) in PBS for 5 minutes.

Live-cell staining was used for the cell-surface marker O4. Live cellswere blocked with 10% normal goat serum and treated with O4 antibody for20 min at 37° C. Cells were gently rinsed three times with warm neuralbase medium and fixed in 4% paraformaldehyde. Samples were rinsed withPBS and incubated with an Alexa Fluor-labeled secondary antibody(Invitrogen, Carlsbad, Calif.; 4 μg ml⁻¹) for 1 hour at roomtemperature. Cells were then permeabilized and stained with DAPI tovisualize nuclei.

Primary antibodies used were antibodies to Oct3/4 (Santa Cruz, sc-5279;0.4 μg ml ⁻¹), Sox1 (R&D Systems, AF3369; 1 μg ml⁻¹), Pax6 (Covance,PRB-278P; 0.67 μg ml⁻1), Olig2 (Millipore, Billerica, Mass., AB9610;1:1,000), Nkx2.2 (DSHB, 74.5A5; 4.4 μg ml⁻¹), Sox10 (R&D Systems,AF2864; 2 μg ml⁻1), O4 (1:10), GFAP (DAKO, Z0334; 0.58 μg ml⁻¹),βIII-tubulin (Covance, Tuj1; 0.2 μg ml⁻¹), and MBP (Covance, SMI-99P; 2μg ml⁻¹ or Abcam, ab7349; 1:25). The Nkx2.2 (74.5A5) antibody developedby T. Jessell and S. Brenner-Morton was obtained from the DevelopmentalStudies Hybridoma Bank developed under the auspices of the US NationalInstitute of Child Health and Human Development and maintained by TheUniversity of Iowa.

For embryo tissue, embryonic day 11.5 (E11.5) embryos were fixed in 4%paraformaldehyde and cryosectioned. After antigen retrieval using sodiumcitrate buffer (10 mM sodium citrate and 0.05% Tween 20; pH 6.0),sections were blocked in 10% donkey serum with 0.2% Triton X for 2hours. Sections were then stained in blocking solution using Olig2(Millipore, Billerica, Mass., AB9610; 1:200) and Nkx2.2 (DSHB, 74.5A5;8.8 μg ml⁻¹) antibodies overnight at 4° C. Alexa Fluor-labeled secondaryantibodies (Invitrogen; 4 μg ml−1) were used for detection, and nucleiwere visualized with DAPI.

For flow cytometry of PDGFRα and NG2, EpiSC-derived OPCs were collectedfrom culture and blocked in 10% normal donkey serum for 30 minutes.Cells were then stained with allophycocyanin (APC)-conjugated PDGFRα(eBioscience, APA5; 4 μg ml⁻¹) and unconjugated rabbit polyclonal NG2(Millipore, Billerica, Mass., AB5320; 1 μg ml⁻¹) antibodies for 30minutes followed by incubation with Alexa Fluor-labeled secondaryantibody (Invitrogen; 4 μg ml⁻¹) for 20 minutes. Isotype controlantibodies were used as a staining control and to set gates(APC-conjugated rat IgG (eBioscience; 4 μg ml⁻¹) and normal rabbit IgG(Santa Cruz; 1 μg ml⁻¹) with Alexa Fluor-labeled secondary antibody(Invitrogen; 4 μg ml⁻¹)). Cells were analyzed on a BD FACSAria, andplots were generated with WinList 3D 7.0 software. Quadrant gates wereset with isotype controls at less than 0.1% double-positive cells.

Example 5 Co-Culture Myelination Analysis

Primary neurons were obtained from the cortex of Ell mice. Followingdissection, the cortex was incubated in 0.125% trypsin in Earle'sbalanced salt solution (EBSS) for 8 minutes at 37° C. and resuspended in10 ml of DMEM containing 10% FBS. The cortex was centrifuged at 600 gfor 2 minutes and resuspended in 1 ml of neural basal medium (NBM)containing 2% B27 (Invitrogen, Carlsbad, Calif.) and 0.25% Glutamax2.The cortex was then triturated through three glass pipettes ofdecreasing diameter, centrifuged, resuspended in supplemented NBM, andfiltered through a 40-μm cell strainer (BD Biosciences, Franklin Lakes,N.J.). Cells were plated on chamber coverslips that had been coated withpoly(d-lysine) and laminin and maintained at 37° C., 10% CO₂ for oneweek. EpiSC-derived OPCs were suspended in 1:1 (vol/vol) mixture ofneurobasal and DMEM (ND) growth medium 2 and added to the neuroncultures. Cells were processed for immunofluorescence after 6 days ofco-culture and analyzed using MBP (Abcam, Cambridge, Mass.) andβIII-tubulin (Tuj1; Neuromics, Edina Minn.) antibodies. Alexafluor-labeled secondary antibodies (2 μg ml⁻¹) were used for detectionand nuclei were visualized with DAPI.

Example 6 In Vivo Mylination Analysis

All experiments with animals were approved by the Institutional AnimalCare and Use Committee of Case Western Reserve University. Earlypostnatal day 1-3 (P1-P3) shiverer (Mbpshi/shi) mice served as a hostfor the transplantation of EpiSC-derived OPC. Pups were anesthetizedwith sofluorane and 2.5×10⁵ cells suspended in 1.5 μl of neural basemedium were injected unilaterally to target the future corpus callosumat a rate of 0.5 μl min⁻¹. Injections were performed with a Hamiltonsyringe through the skull +0.5 mm right of midline and +0.5 mm anteriorto bregma at a depth of 2 mm. Mice injected with EpiSC-derived OPCs aswell as controls were killed at various time points to analyzemyelination. Mice were deeply anaesthetized with rodent cocktail(ketamine, xylazine and acepromazine), perfused transcardially with 0.9%saline at room temperature and then perfused with ice-cold 4%paraformaldehyde. The brain was dissected and fixed for 2 hours in 4%paraformaldehyde then cryoprotected with sucrose. The cortex was frozenin optimal cutting temperature (OCT) medium on dry ice and 20 μmsections were cut on a Microm 525 cryostat. The sections were air-driedand then frozen. For fluorescent MBP staining, slides were thawed andallowed to dry, then rehydrated in PBS. Slides were treated with icecold 95% methanol and 5% acetic acid for 7 minutes, rinsed in PBS andblocked in 10% goat serum for 1 hours at room temperature. Slides werethen stained overnight for MBP (Covance; 2 μg ml⁻¹). Alexa Fluor-labeledsecondary antibodies (2 μg ml⁻¹) were used for detection and nuclei werevisualized with DAPI. Stained sections were mounted using Vectashield(Vector Labs) and imaged using a Zeiss LSM 510 META laser scanningconfocal microscope. All images presented are maximum intensityprojections of a z-dimension series consisting of 1.8-μm optical slicescollected every 0.9 μm (optimal interval setting determined by LSM 510software).

Example 7 Organotypic Slice Culture Myelination Analysis

The forebrain of P2-P4 shiverer or wild-type mice was dissected and 300μm coronal slices were made on a Leica Vibratome. Slices were culturedas previously described in a DMEM-Basal Medium Eagle's base with 15%horse serum, modified N2 and PDGF-AA for 3 days. 2×10⁵ EpiSC-derivedOPCs was manually injected with a pulled glass pipette into the slicesand grown for an additional 10 days in culture. Some OPC cultures werefirst lentivirally labeled with EGFP. Slices were fixed in 4%paraformaldehyde, treated with ice-cold 95% methanol and 5% acetic acid,and assayed for MBP expression (Covance; Jackson Labs, biotin-anti-mouseIgG; Vector Labs, ABC; Sigma, DAB). For lineage-tracing experiments,slices were assayed for NeuN expression (Millipore, Billerica, Mass.,ABN78) and GFP (Invitrogen, Carlsbad, Calif. 3E6). To analyze the myelinultrastructure, fixed and MBP-stained slices were then fixed (4%paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer, pH7.4), incubated in 1% osmium tetroxide and stained en bloc in uranylacetate. They were dehydrated and embedded in Poly/Bed 812 epoxy. Thick(1 μm) sections were cut and stained with toluidine blue. Thin (90 nm)sections were cut either en face or transversely, collected on 300-μmnickel grids, stained with toluidine blue and carbon-coated for electronmicroscopy. Toluidine blue-stained thick sections were imaged on a LeicaDM5500B microscope at 100×, and toluidine blue-stained thin sectionswere imaged at 80 kV on a JEOL JEM-1200-EX electron microscope. Stainingof sections for MBP before electron microscopy allowed for efficienttargeting of the regions to be analyzed.

Example 7 Results

To differentiate epiblast stem cells (EpiSCs) into a neuroectodermallineasge, EpiSCs were treated with SB431542, an inhibitor of Alk4, Alk5and Alk7 (activin-nodal signaling); dorsomorphin or LDN-193189,inhibitors of Alk2, Alk3 and Alk6 (BMP signaling); and noggin, a BMPantagonist. In response, the EpiSCs rapidly downregulated the expressionof pluripotency genes such as Pou5f1 (also known as Oct3/4), upregulatedneuroectodermal genes such as Pax6 and Neurod4, and underwentmorphological changes to form radially-organized patterned neuroectoderm(FIG. 2). This differentiation strategy was extremely robust andresulted in >99% of EpiSC colonies forming patterned neuroectodermexpressing both Pax6 and Sox1 (Pax6+ and Sox1+).

To test whether the induced patterned neuroectoderm behaved asun-induced neuroectoderm behaves, the induced EpiSC-derived patternedneuroectoderm were patterned in a region-specific manner. In thedeveloping neural tube, specific cellular precursor domains areestablished by local signals from the surrounding tissues. In thedeveloping spinal cord, OPCs first emerge from the ventral ventricularzone of the neural tube in response to sonic hedgehog (SHH) and othersignals from the notochord and floor plate. Day-4 EpiSC-derivedpatterned neuroectoderm was treated for 1 day with specificconcentrations of retinoic acid and SHH in the continued presence ofnoggin to pattern them in a region-specific way. This treatment resultedin the upregulation of OPC-relevant transcription factors Olig2 andNkx2.2. The expression pattern of these factors in the patternedneuroectoderm mimicked the non-overlapping expression in the ventralventricular zone of the developing mouse neural tube. Additionally, theEpiSC-derived patterned neuroectoderm expressed proper posterior-relatedHox genes, thus confirming their spinal cord identity along therostrocaudal axis. These results demonstrate that the provision ofguiding developmental cues leads to proper specification and patterningof pluripotent cells into region-specific cells of the developing spinalcord in 5 days.

Upon passage, the EpiSC-derived patterned neuroectoderm immediately gaverise to both βIII-tubulin+ neurons and presumptive OPCs that expressedOlig2 or Nkx2.2. These results recapitulate in vivo cell-specificationevents at the ventral ventricular zone of the developing spinal cordthat are known to give rise to both neurons and OPCs. In these cultures,the neurons did not persist beyond the first 3 days of culture. However,the presumptive OPCs developed strong, co-expression of Olig2 and Nkx2.2and rapidly proliferated in the presence of PGF2, PDGF and SHH toproduce a confluent and nearly homogeneous population of cells in 5 days(10 days from initial differentiation of the EpiSCs). TheseEpiSC-derived OPCs exhibited a typical bipolar morphology and expressedtranscription factors and cell surface markers such as Olig1, Olig2,Nkx2.2, Sox10, PDGFRα, EGFR and NG2 (Cspg4) consistent with their invivo counterparts (FIG. 3). The EpiSC-derived OPCs were synchronous andhighly pure as ˜90% co-expressed the OPC surface markers NG2 and PDGFRα,as determined by flow cytometry, without selection or sorting.Additionally, these EpiSC-derived OPCs could be expanded for at leasteight passages, yielding previously unobtainable numbers (>10¹² cells)of pure OPCs. To demonstrate the robust nature of this method, fourindependently-derived EpiSC lines (including lines from mice ofdifferent genders, strains and developmental stage) were simultaneouslydifferentiated. All four cell lines differentiated efficiently into OPCswithout any cell line-specific modifications.

To determine the differentiation capacity of the EpiSC-derived OPCs, theEpiSc-derived OPCs were treated with thyroid hormone (T3), which isknown to be important both in vitro and in vivo in regulating thetransition of OPCs into oligodendrocytes, in the absence of both FGF2and PDGF-AA. These conditions caused the EpiSC-derived OPCs to stopproliferating and differentiate exclusively into oligodendrocytes over a2-4 day time course. Undifferentiated OPCs did not express theoligodendrocyte cell-surface marker O4, but by day 2 of differentiationgreater than 64% of the cells were strongly O4+ with a multiprocessedmorphology and the rest remained Olig2+ OPCs (cells manually countedfrom random fields of independent lines, n>480 cells). By day 3, theseO4+ oligodendrocytes had upregulated the classical and defining markersof bona fide oligodendrocytes such as myelin basic protein (Mbp),proteolipid protein 1 (P1p1), myelin-associated glycoprotein (Mag) andmyelin oligodendrocyte glycoprotein (Mog) (FIG. 4). The differentiationwas highly specific as neither GFAP+ astrocytes or βIII-tubulin+ neuronswere observed. By day 4, the overwhelming majority of EpiSC-derived OPCshad become oligodendrocytes, though cell death and rare proliferationevents made exact quantification challenging without large-scaletime-lapse lineage analysis. To test the robust nature of this protocol,OPCs derived from four independent EpiSC lines were differentiated intooligodendrocytes, and there were no apparent differences in theefficiency or timing of differentiation. Freezing-thawing and extensiveexpansion of the EpiSC-derived OPC cultures did not alter theirproperties or ability to differentiate as all passages tested hadhighly-correlated global gene expression patterns and generatedoligodendrocytes with similar timing and efficiency.

To examine the functional properties of EpiSC-derived OPCs, myelinogenicpotential was measured by in vitro culture with neurons as well as inorganotypic slice culture and in vivo by injection into the brains ofcongenitally hypomyelinated mice. For in vitro culture studies,EpiSC-derived OPCs were plated at low density onto cultures of mousecortical neurons and allowed to differentiate. After 6 days, theEpiSC-derived OPCs had differentiated into MBP+ cells. Much of the MBP+staining aligned with βIII-tubulin+ axons, which is suggestive ofmyelinogenic capacity. EpiSC-derived OPCs previously exposed to T3 didnot produce MBP+ segments, which tracked with βIII-tubulin+ cells,suggesting temporal restriction of myelinogenic potential. To assessfurther the functionality of the EpiSC-derived OPCs, a tractableorganotypic slice culture assay was utilized to evaluate myelinogenicpotential. Injection of EpiSC-derived OPCs into coronal, forebrainslices of early postnatal shiverer (Mbpshi/shi) pups (which lack Mbp andcompact myelin) resulted in substantial numbers of Mbp+ segments andcompact myelin after 10 days. Transplanted EpiSC-derived OPCsspecifically differentiated into oligodendrocytes and did not formneurons in the shiverer brain slices. The ability of EpiSC-derived OPCsto myelinate shiverer host axons in slice culture using both low- andhigh-passage number cells was assessed, with no evident differences.

To assess the myelinogenic capacity of EpiSC-OPCs in vivo, 2.5×10⁵ cellswere delivered into the future corpus callosum of newborn,immunocompetent shiverer (Mbpshi/shi) pups. The mouse brains wereanalyzed 3-7 weeks after injection and assayed for the presence ofmyelinated fibers in the brain. Many patches of MBP+ myelin sheaths werefound in mice transplanted with EpiSC-derived OPCs but not inuntransplanted controls. Additionally, EpiSC-derived OPCs appeared tomigrate extensively in the host central nervous system as myelination atsites distant from the injection such as the contralateral striatum wasobserved. No teratomas or aberrant cellular growths were observed in anyof the mice transplanted with EpiSC-derived OPCs at the time pointsevaluated. Taken together, these results demonstrate that EpiSC-derivedOPCs function to produce compact myelin and provide a tractable sourceof cells to optimize cell-based transplantation strategies for myelinrepair.

Next, the ability of extrinsic factors to modulate the differentiationof Epi-SC-derived OPCs was explored. As this invention allows for thescalable production of OPCs from pluripotent cells, it provides aplatform to screen for molecules that influence the transition of OPCsinto oligodendrocytes; particularly pertinent to disorders such asmultiple sclerosis where NG2+ OPCs are present in or close todemyelinated lesions but generally do not remyelinate demyelinatedaxons. As a simple proof-of-principle for using the system to screen forcompounds, EpiSC-derived OPCs were exposed to three different treatmentregimens involving modulation of signaling pathways (Notch,Wnt-β-catenin and BMP) previously implicated in oligodendrocytedevelopment or differentiation. Treatment of EpiSC-derived OPCs for 2days with T3 differentiation medium in the presence of the Notch ligandJagged1 (Jag1) did not impact the rate or number of O+ oligodendrocytes.Using a similar protocol, the role of GSK3β, a negative regulator ofWnt-β-catenin signaling, was tested by treating EpiSC-derived OPCs withthe GSK3β inhibitor CHIR99021. This treatment resulted in aconcentration-dependent inhibition of oligodendrocyte differentiationwhereby cells remained Olig2+ OPCs and did not progress to O4+oligodendrocytes (FIG. 5). These results suggest that canonicalWnt-β-catenin signaling, activated by inhibition of GSK3β, positivelyregulates the OPC state and blocks the transition to oligodendrocytes.

The final treatment involved exposure of EpiSC-derived OPCs to signalingcascades known to respecify OPCs in vitro into an alternate glial fate,astrocytes. Treatment of EpiSC-derived OPCs with BMP4 and leukemiainhibitory factor (LIF) resulted in the majority of cells undergoingmorphological and gene expression changes including the upregulation ofGFAP, indicative of respecification into astrocytes. These resultsdemonstrate that scalable mouse pluripotent stem cell-based systemprovides a powerful way to screen for modulators of oligodendrocytedevelopment and myelination.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryaspects shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims. In the claims thatfollow, unless the term “means” is used, none of the features orelements recited therein should be construed as means-plus-functionlimitations pursuant to 35 U.S.C. §112, ¶6.

What is claimed is:
 1. A method for generating mammalian neuroectodermcomprising: providing pluripotent cells; and inducing development of thepluripotent cells into neuroectoderm by culturing the pluripotent cellsin the presence of one or more inhibitors of an activin-nodal pathwayand one or more inhibitors of a bone morphogenetic protein pathway. 2.The method of claim 1, wherein the one or more inhibitors of anactivin-nodal pathway is SB431542.
 3. The method of claim 1, wherein theone or more inhibitors of a bone morphogenetic protein pathway isselected from dorsomorphin, LDN-193189 or noggin.
 4. The method of claim1, wherein the pluripotent cell is an epiblast stem cell.
 5. The methodof claim 1, wherein at least 75% or more of cells resulting from theculture of the pluripotent cells are neuroectoderm cells.
 6. The methodof claim 5, wherein at least 90% or more of cells resulting from theculture of the pluripotent cells are neuroectoderm cells.
 7. A methodfor generating mammalian patterned neuroectoderm comprising: providingpluripotent cells; inducing development of the pluripotent cells intoneuroectoderm by culturing the pluripotent cells in the presence of oneor more inhibitors of an activin-nodal pathway and one or moreinhibitors of a bone morphogenetic protein pathway; and inducingdevelopment of patterned neuroectoderm by culturing the neuroectoderm inthe presence of one or more of sonic hedgehog, retinoic acid and noggin.8. A method for generating mammalian oligodendrocyte progenitor cellscomprising: providing a pluripotent cell; inducing development of thepluripotent cell into neuroectoderm by culturing the pluripotent cell inthe presence of one or more inhibitors of an activin-nodal pathway andone or more inhibitors of a bone morphogenetic protein pathway; inducingdevelopment of patterned neuroectoderm by culturing the neuroectoderm inthe presence of one or more of sonic hedgehog, retinoic acid and noggin;and inducing development of oligodendrocyte progenitor cells byculturing the patterned neuroectoderm cells in the presence of one ormore of a fibroblast growth factor, a platelet-derived growth factor andsonic hedgehog.
 9. The method of claim 8, wherein two or more of afibroblast growth factor, a platelet-derived growth factor and sonichedgehog are used in the culture of the patterned neuroectoderm cells.10. The method of claim 9, wherein a fibroblast growth factor, aplatelet-derived growth factor and sonic hedgehog are used in theculture of the patterned neuroectodermal cells.
 11. The method of claim8, wherein the fibroblast growth factor is PGF2 and the platelet-derivedgrowth factor is PDGF-AA.
 12. The method of claim 8, wherein two ofretinoic acid , sonic hedgehog and noggin are used in the culture of theneuroectodermal cells.
 13. The method of claim 8, wherein retinoic acid,sonic hedgehog and noggin are used in the culture of the neuroectodermalcells.
 14. The method of claim 8, wherein at least 75% or more of cellsresulting from the culture of the patterned neuroectodermal cells areoligodendrocyte progenitor cells.
 15. The method of claim 14, wherein atleast 90% or more of cells resulting from the culture of the patternedneuroectodermal cells are oligodendrocyte progenitor cells.
 16. Themethod of claim 15, wherein at least 95% or more of cells resulting fromthe culture of the patterned neuroectodermal cells are oligodendrocyteprogenitor cells.
 17. The method of claim 16, wherein at least 99% ormore of cells resulting from the culture of the patternedneuroectodermal cells are oligodendrocyte progenitor cells.
 18. Themethod of claim 8, wherein the one or more inhibitors of anactivin-nodal pathway is SB431542.
 19. The method of claim 8, whereinthe one or more inhibitors of a bone morphogenetic protein pathway isselected from dorsomorphin, LDN-193189 or noggin.
 20. A compositioncomprising the oligodendrocyte progenitor cells produced according toclaim
 8. 21. The composition of claim 20, further comprising neurons orneuron precursors.
 22. A research tool comprising the oligodendrocyteprogenitor cells produced according to claim
 8. 23. A diagnostic toolcomprising the oligodendrocyte progenitor cells produced according toclaim
 8. 24. A method of treating a medical condition of the CNS in asubject comprising administering to the subject a therapeuticallyeffective amount of the oligodendrocyte progenitor cells producedaccording to claim
 8. 25. A method for maintaining the oligodendrocyteprogenitor cells of claim 8 in cell culture, comprising: culturing theoligodendrocyte cells in the presence of an activator of Wnt-β-cateninsignaling.
 26. A method for maintaining the oligodendrocyte progenitorcells of claim 8 in cell culture, comprising: culturing theoligodendrocyte cells in the presence of fibroblast growth factor (FGF),platelet-derived growth factor (PDGF) and sonic hedgehog (SHH).
 27. Themethod of claim 26, wherein at least 95% of cells in culture areoligodendrocyte progenitor cells.
 28. The method of claim 27, wherein atleast 99% of cells in culture are oligodendrocyte progenitor cells. 29.The method of claim 28, wherein the activator of Wnt-β-catenin signalingis a GSK3β inhibitor.
 30. The method of claim 29, wherein the GSK3βinhibitor is CHIR99021.
 31. A composition comprising the oligodendrocyteprogenitor cells maintained according to claim
 25. 32. The compositionof claim 31, further comprising neurons or neuron precursors.
 33. Aresearch tool comprising the oligodendrocyte progenitor cells maintainedaccording to claim
 25. 34. A diagnostic tool comprising theoligodendrocyte progenitor cells maintained according to claim
 25. 35. Amethod of treating a medical condition of the CNS in a subjectcomprising administering to the subject a therapeutically effectiveamount of oligodendrocyte progenitor cells maintained according to claim25.
 36. A method for generating oligodendrocytes from theoligodendrocyte progenitor cells maintained according to claim 25,comprising culturing the oligodendrocyte progenitor cells in the absenceof FGF and PDGF and presence of T3.
 37. The method of claim 36, wherethe oligodendrocyte differentiation activator is thyroid hormone.
 38. Amethod for generating astrocytes from the oligodendrocyte progenitorcells maintained according to claim 25, comprising culturing themaintained oligodendrocyte progenitor cells in the presence of a bonemorphogenetic protein and a JAK/STAT pathway activator.
 39. The methodof claim 38, wherein the bone morphogenetic protein is bonemorphogenetic protein 4 and the JAK/STAT pathway activator is leukemiainhibitory factor.
 40. A method for generating mammalian oligodendrocyteprogenitor cells and maintaining the mammalian oligdendrocyte progenitorcells in culture comprising: providing pluripotent cells; inducingdevelopment of the pluripotent cells into neuroectoderm by culturing thepluripotent cells in the presence of one or more inhibitors of anactivin-nodal pathway and one or more inhibitors of a bone morphogeneticprotein pathway; inducing development of patterned neuroectoderm byculturing the neuroectoderm in the presence of sonic hedgehog, retinoicacid and noggin; inducing development of oligodendrocyte progenitorcells by culturing the patterned neuroectoderm cells in the presence ofa fibroblast growth factor, a platelet-derived growth factor and sonichedgehog; and maintaining the oligodendrocyte progenitor cells culturingthe oligodendrocyte cells in the presence of an activator ofWnt-β-catenin signaling.
 41. The method of claim 40, wherein the one ormore inhibitors of an activin-nodal pathway is SB431542 and the one ormore inhibitors of a bone morphogenetic protein pathway is selected fromdorsomorphin, LDN-193189 or noggin.
 42. The method of claim 40, whereinthe fibroblast growth factor is PGF2 and the platelet-derived growthfactor is PDGF-AA.
 43. The method of claim 40, wherein at least 95% ofcells in culture are oligodendrocyte progenitor cells.
 44. A method forgenerating patterned neuroectoderm from neuroectoderm comprisingculturing the neuroectoderm in the presence of sonic hedgehog, retinoicacid and noggin.
 45. A method for generating oligodendrocyte progenitorcells from patterned neuroectoderm comprising culturing the patternedneuroectoderm cells in the presence of a fibroblast growth factor, aplatelet-derived growth factor and sonic hedgehog.
 46. A method formaintaining oligodendrocyte progenitor cells in cell culture,comprising: culturing the oligodendrocyte cells in the presence of anactivator of Wnt-β-catenin signaling.
 47. A method for generatingastrocytes from oligodendrocyte progenitor cells, comprising culturingthe maintained oligodendrocyte progenitor cells in the presence of abone morphogenetic protein and a JAK/STAT pathway activator.
 48. Amethod for generating oligodendrocytes from oligodendrocyte progenitorcells maintained, comprising culturing the oligodendrocyte progenitorcells in the absence of FGF and PDGF and presence of T3.